Bioinformatic and structure/function studies suggest that most extant metabolic pathways were patched together from previously existing promiscuous enzyme activities that, even if inefficient, provided some degree of catalysis. However, we cannot explain why certain pathways arose rather than the thousands of other possibilities. Further, we have little insight into the process by which novel pathways were patched together and flux was improved via mutations. This project will exploit a model system in which the pathway for synthesis of pyridoxal 5- phosphate (PLP) has been disrupted to identify ?serendipitous pathways? (SPs) that restore PLP synthesis by patching together promiscuous activities of enzymes that normally serve other functions. The enzymes that catalyze steps in SPs will be characterized. The mechanisms by which mutations increase flux through a SP to a physiologically significant level will be identified. Finally, the dependence of the evolutionary outcome on environmental conditions and genome content will be defined. The feasibility of this project is supported by preliminary results identifying two SPs after evolution of ?pdxB E. coli in different conditions and evidence that PLP synthesis can be restored after evolution in other conditions, as well. Biochemical, genetic, and metabolomics approaches have revealed the mechanisms by which mutations enabled assembly of one of these SPs. E. coli and other bacteria in which a gene required for PLP synthesis has been deleted will be subjected to laboratory evolution. If growth can be restored, the responsible mutations will be identified. The mechanisms by which these mutations have facilitated recruitment of a promiscuous enzyme to replace the missing enzyme, or, more interestingly, emergence of a SP will be defined using a battery of biochemical, genetic and ?omics approaches. This project will provide new insights into the assembly of metabolic pathways, how genome content and growth conditions affect the evolution of novel pathways, and how flux through initially inefficient pathways can be elevated due to mutations. These insights will help us understand how bacteria have evolved throughout the history of life on earth, and how they will continue to evolve as humans exert new selective pressures due to introduction of anthropogenic chemicals such as pesticides and pharmaceuticals. Evolution of pathways capable for degradation of anthropogenic compounds is critical to minimize adverse effects on the health of ecosystems and humans. On the other hand, evolution of novel pathways might be an unrecognized mechanism for resistance to antimetabolite drugs used to treat infectious diseases and cancer.